Abstract:

Provided are methods, kits and arrays for use in determining whether a
scar of interest is keloid or non-keloid in nature. These determine
keloid or non-keloid nature based on comparison of gene expression in the
scar of interest with expression in a control sample. If expression of at
least one gene, selected from the group of genes set out in Table 1, is
decreased in a sample representative of gene expression in the scar of
interest compared to expression of the same gene (or genes) in the
control sample this indicates that the scar of interest comprises a
keloid.

Claims:

1. A method for diagnosing a scar of interest as keloid or non-keloid, the
method comprising: comparing expression in a sample representative of
gene expression in the scar of interest, of at least one gene, selected
from the group of genes set out in Table 1, with expression of the said
at least one gene in a comparator tissue; wherein decreased expression of
said at least one gene in the scar of interest compared to expression of
said at least one gene in the comparator tissue indicates that the scar
of interest comprises a keloid.

2. A method according to claim 1, wherein the method is an in vitro
method.

3. A method according to claim 1, comprising comparing the expression of
at least one gene selected from the group of genes set out in Table 2.

4. A method according to claim 1, comprising comparing the expression of
at least one gene selected from the group of genes set out in Table 3.

5. A method according to claim 1, comprising comparing the expression of
at least one gene selected from the group of genes set out in Table 8.

6. A method according to claim 1 comprising comparing the expression of at
least one gene selected from the group of genes set out in Table 13.

7. A method according to claim 1, comprising comparing the expression of
at least one gene selected from the group of genes set out in Table 17.

8. A method according to claim 1, comprising comparing the expression of
at least one gene selected from the group of genes set out in Table 20.

9. A method according to claim 1, comprising comparing the expression of
at least one gene selected from the group of genes set out in Table 21.

10. A method according to claim 1 comprising comparing the expression of
at least one gene selected from the group of genes set out in Table 22.

11. A method according to claim 1, comprising comparing the expression of
at least one gene selected from the group of genes set out in Table 24.

12. A method according to claim 1, comprising comparing the expression of
at least one gene selected from the group of genes set out in Table 26.

13. A method according to claim 1, wherein the sample representative of
gene expression in the scar of interest comprises a nucleic acid target
molecule.

16. A method according to claim 1, wherein the sample representative of
gene expression in the scar of interest comprises a protein target
molecule.

17. A method according to claim 13, wherein the comparison of gene
expression is effected using a probe molecule capable of binding
specifically to the target molecule.

18. A method according to claim 17, wherein the probe molecule is selected
from the group comprising oligonucleotide probes, antibodies and
aptamers.

19. A method according to claim 1, wherein expression. in the sample and
expression in the comparator tissue is compared for at least 5 genes.

20. A method according to claim 1, wherein expression in the sample and
expression in the comparator tissue is compared for between 5 and 10
genes.

21. A kit for diagnosing a scar of interest as keloid or non-keloid, the
kit comprising: i) at least one probe capable of binding specifically to
a target molecule representative of expression in the scar of interest of
at least one gene selected from the group set out in Table 1; and ii)
reference material able to indicate the level of expression of said at
least one gene in comparator tissue.

22. A kit according to claim 21, wherein the probe comprises an
oligonucleotide probe.

23. A kit according to claim 21, wherein the probe comprises an antibody.

24. A kit according to claim 21, wherein the probe comprises an aptamer.

25. A kit according to claim 21, wherein the probe is a labelled probe.

26. A kit according to claim 25, wherein the probe is a
fluorescent-labelled probe.

27. A kit according to claim 25, wherein the probe is an enzyme-labelled
probe.

28. A kit according to claim 25, wherein the probe is a
radioactive-labelled probe.

29. A kit according to claim 21, comprising probes capable of binding
specifically to target molecules representative of expression of at least
5 genes selected from the group set out in Table 1.

30. A kit according to claim 21, comprising probes capable of binding
specifically to target molecules representative of expression of between
5 and 10 genes selected from the group set out in Table 1.

31. A kit according to claim 21, wherein the kit comprises probes capable
of binding specifically to target molecules representative of gene
expression of at least one gene selected from those set out in Table 2;
and/or those set out in Table 3; and/or those set out in Table 8; and/or
those set out in Table 13; and/or those set out in Table 17 and/or those
set out in Table 20; and/or those set out in Table 21; and/or those set
out in Table 22; and/or those set out in Table 24; and/or those set out
in Table 26.

32. A kit according to claim 21, wherein the reference material comprises
a library of nucleic acid targets representative of expression of said at
least one gene selected from the group of genes set out in Table 1.

33. A kit according to claim 21, wherein the reference material comprises
a library of protein targets representative of expression of said at
least one gene selected from the group of genes set out in Table 1.

34. A kit according to claim 21, wherein the reference material comprises
data as to the expression of said at least one gene selected from the
group of genes set out in Table 1.

35. A kit according to claim 21, further comprising a diagnostic
algorithm.

36. A kit according to claim 21, further comprising assay control material
able to indicate that an assay has been performed correctly.

37. A kit according to claim 21, further comprising materials for the
preparation of a population of target molecules representative of gene
expression in a scar of interest.

38. An array of oligonucleotide probes, characterised in that at least
7.0% of the oligonucleotides probes present in the array are selected
from the group of genes set out in Table l.

39. An array comprising a nylon substrate to which are adhered nucleic
acid probes representative of genes selected from the group of genes set
out in Table 1.

40. An array comprising immobilized antibody probes capable of binding
specifically to molecules representative of expression of one or more of
the group of genes set out in Table l.

41. An array according to claim 38, wherein the array comprises probes
capable of binding specifically to target molecules representative of
gene expression of at least one gene selected from those set out in Table
2; and/or those set out in Table 3; and/or those set out in Table 8;
and/or those set out in Table 13; and/or those set out in Table 17 and/or
those set out in Table 20; and/or those set out in Table 21; and/or those
set out in Table 22; and/or those set out in Table 24; and/or those set
out in Table 26.

42. An array according to claim 39, wherein the array comprises probes
capable of binding specifically to target molecules representative of
gene expression of at least one gene selected from those set out in Table
2; and/or those set out in Table 3; and/or those set out in Table 8;
and/or those set out in Table 13; and/or those set out in Table 17 and/or
those set out in Table 20; and/or those set out in Table 21; and/or those
set out in Table 22; and/or those set out in Table 24; and/or those set
out in Table 26.

43. An array according to claim 40, wherein the array comprises probes
capable of binding specifically to target molecules representative of
gene expression of at least one gene selected from those set out in Table
2; and/or those set out in Table 3; and/or those set out in Table 8;
and/or those set out in Table 13; and/or those set out in Table 17 and/or
those set out in Table 20; and/or those set out in Table 21; and/or those
set out in Table 22; and/or those set out in Table 24; and/or those set
out in Table 26.

44. A method according to claim 16, wherein the comparison of gene
expression is effected using a probe molecule capable of binding
specifically to the target molecule.

Description:

[0001]The invention relates to a method of diagnosing a scar of interest
as keloid or non-keloid. The invention also provides kits and
oligonucleotide arrays suitable for use in the diagnosis of a scar of
interest as keloid or non-keloid.

[0002]Keloids (also referred to as keloid scars) are pathological scars
produced by an aberrant and over-exuberant wound healing response.
Keloids comprise raised scars that spread beyond the margins of an
original wound and invade the normal skin surrounding the wound site.
Keloids continue to grow over time, and do not regress spontaneously.

[0003]Keloids occur with equal frequency in men and women. The incidence
of keloid formation is increased in those aged between 10 and 30 years.
Keloids may arise as a result of a wide range of injuries, including
piercing, surgery, vaccination, tattoos, bites, blunt trauma and burns.

[0004]Keloids may have a "domed", nodular or ridged appearance. Keloids
may have a colour similar to that of the surrounding unwounded skin, but
are frequently somewhat darker, with a red, purple or brown appearance.
Such colour mismatches may increase the visual prominence of keloids. The
tendency for hyperpigmentation in keloids is increased on their exposure
to solar ultraviolet radiation.

[0005]A keloid lesion may be considered to be made up of a number of
different portions that may each exhibit quite different biological
activity from one another. The central part of a mature keloid lesion
(the intra-lesional portion) is largely acellular, while the peripheral
part of the lesion (the peri-lesional portion) is relatively more
cellular and is the site of increased angiogenic activity. This increase
in new blood vessel formation has been linked with the outward growth of
the lesion.

[0006]Although they represent examples of pathological scarring, keloids
are primarily composed of the same cell types and extracellular matrix
components that are found in undamaged skin and normal dermal scars.
However, the relative abundance and arrangement of these cell types and
extracellular matrix components differ from those found in either
unwounded skin or normal dermal scars.

[0007]The major constituent of keloids is the extracellular matrix
component collagen I. Fibroblasts derived from keloids exhibit up to a
twenty-fold higher expression of collagen I in vitro, as compared to
normal dermal fibroblasts. Similarly, cultured keloid fibroblasts also
express elevated levels of elastin and proteoglycans, and it is believed
that this increase in extracellular matrix deposition may play a role in
keloid development and maintenance.

[0008]Collagen I present in keloids is arranged primarily in the form of
thick "whorls", which may be differentiated from the arrangement found in
unwounded skin (a so-called "basket weave" of fibrils) and in normal
scars (which contain collagen fibres that are thinner than those found in
keloids and are arranged approximately parallel to one another). The
frequent presence of thickened hyalinized collagen within keloids has led
to this form of collagen being termed "keloidal collagen".

[0010]Keloids are seldom a direct cause of pain, but may give rise to
discomfort, tenderness, irritation or itching during their formation or
growth. Keloids may also impair mechanical function through their size or
their increased stiffness compared to unwounded skin. This impairment may
be particularly noticeable in the case of keloids located near a joint.
Furthermore, it is well recognised that keloids, and in particular large
or noticeably disfiguring examples, can cause psychological distress to
those afflicted.

[0011]A further highly damaging property of keloids is their propensity to
recur, particularly following surgical excision. Recurrence of keloids
under such circumstances is normally also associated with further
expansion of the lesion, and keloids may expand more aggressively
following an earlier excision.

[0012]Treatment options for hypertrophic scars are similar to those for
keloids with the exception that surgical excision is an acceptable and
often more favourable approach.

[0013]It will, be appreciated that in the case of keloids, it will
generally be preferred to avoid surgical intervention when possible.
Given their high incidence of recurrence, and the fact that such
recurrence is exacerbated by surgical intervention, it is important to be
able to accurately diagnose keloids in order that suitable treatment
regimes may be employed. Current treatment regimes for keloids include
corticosteroid injections, cryotherapy, radiation therapy, silicone gel
dressings and intra-lesional injection of agents intended to reduce the
size of keloid scarring.

[0014]In present practice diagnosis of keloids is undertaken on the basis
of the appearance of the scar. However, the accurate diagnosis of keloids
is hampered by the fact that keloid morphology may be very similar to
that of other pathological scars. The appearance of keloids and
hypertrophic scars may be particularly similar. Hypertrophic scars
resemble keloid scars in that they are also raised above the skin level.
However, hypertrophic scars differ from keloids in that they remain
within the boundaries of the original lesion, and may regress
spontaneously several months after the initial injury. The visual
similarity between keloid and hypertrophic scars means that diagnosis of
a raised scar between these two distinct conditions is often confusing
and cannot be accurately undertaken without long-term monitoring. There
is a need for rapid and accurate means by which scars of interest may be
diagnosed to indicate whether they are keloid in nature, or whether they
belong to other pathological or excessive scarring types, such as
hypertrophic scars.

[0015]Raised scars may often be assumed to be associated with keloid
disease, and in the case of black patients an elevated scar will often be
diagnosed by default as keloidal by many physicians (Rosenborough et al,
2004. J. Natl. Med. Assoc. 96, 108). This tendency can lead to the
mis-identification of non-keloids scars (such as hypertrophic scars or
very bad non-pathological scars) as keloids. It will be appreciated that
this potential mis-diagnosis can result in inappropriate scar management
decisions and can block the use of elective/scar revision surgery as a
viable therapeutic approach in the case of such mis-diagnosed scars.

[0016]It is known that keloids are the only pathological dermal scars that
grow beyond the boundaries of the original injury, as noted above. This
property can provide a basis on which differential diagnosis between
keloid and hypertrophic scars may be undertaken, although such diagnosis
requires a very long time, given the need for prolonged observation of
the scar to be diagnosed.

[0017]Other attempts to provide a basis on which tissues may be diagnosed
as keloid or non-keloid have utilised histological assessments. Among the
histological features suggested as providing suitable basis for diagnosis
of keloids is the presence of so called "keloidal collagen", a thickened
hyalinized form of collagen, although this is not found in all keloid
samples. Further features that may allow for the differentiation of
keloids from other pathological scars (such as hypertrophic scars) are
the presence of a non-flattened epidermis in keloids, non-fibrotic
papillary dermis, the presence of a "tongue-like" advancing edge that
surrounds keloid lesions (located below the normal-appearing epidermis
and papillary dermis), presence of a horizontal cellular fibrous band
located in the upper reticular dermis, and the presence of a prominent
fascia-like band.

[0018]However, these histological cues for the diagnosis of keloids are
also unsatisfactory. Not all of the features suggested as diagnostic
markers are found in all keloid tissues, and similarly some of the
suggested markers may also be found in non-keloid tissues.

[0019]Furthermore, the use of histological means for diagnosis of keloids
requires considerable time to be expended in the preparation and analysis
of histological samples, as well as the need for the application of skill
and judgement on the part of the person undertaking such analysis.

[0020]Rapid and accurate methods and kits for the diagnosis of keloid
scars will enable diagnosis to be undertaken with greater confidence.
This will facilitate taking of correct decisions regarding the clinical
treatment of skin lesions, and will be advantageous in treatment of both
keloid and non-keloid lesions. In the case of tissues diagnosed as
keloids it will be possible to avoid treatments that may otherwise
exacerbate keloid recurrence and expansion, while such considerations
will not be inappropriately applied in the treatment of non-keloid
tissues. Furthermore, it is likely that early accurate diagnosis may have
major benefits in relation to the success of palliative care regimes
treatments, since many available treatments are believed to be more
effective on less-mature scars.

[0021]The ability to differentiate between keloid-forming and
non-keloid-forming patients may thus provide great advantages in terms of
limiting surgery, and hence the risk of keloid formation, amongst those
prone to keloid development, since it is generally considered that the
prevention of keloid formation is of paramount importance in the
management of keloid-forming patients, and avoidance of non-essential
cosmetic surgery is recommended for these individuals.

[0022]In the light of the above it will be appreciated that there exists a
well recognised need for the provision of new and alternative methods and
kits for the diagnosis of keloids. Such methods and kits may preferably
be suited to the safe and reliable diagnosis of keloids.

[0023]It is an aim of certain embodiments of the invention to provide
novel methods and markers for the diagnosis of keloids. It is another aim
of certain embodiments of the present invention to provide methods of
diagnosis that allow a greater degree of certainty in diagnosis of a scar
of interest as keloid or non-keloid than may be achieved by the prior
art. It is another aim of certain embodiments of the invention to provide
methods of diagnosis that allow greater speed of diagnosis, to determine
whether a scar of interest is keloid or non-keloid, than do the methods
of the prior art. It is still another aim of certain embodiments of the
invention to provide methods for the diagnosis of a scar of interest as
keloid or non-keloid that do not require the taking of large biopsies in
order for a diagnosis to be made. It is still another aim of certain
embodiments of the invention to provide methods, allowing the diagnosis
of a scar of interest as keloid or non-keloid, that do not involve
procedures that are likely to cause the recurrence and/or exacerbation of
keloid formation.

[0024]According to a first aspect of the present invention there is
provided a method for diagnosing a scar of interest as keloid or
non-keloid, the method comprising:

comparing expression in a sample representative of gene expression in the
scar of interest of at least one gene, selected from the group of genes
set out in Table 1, with expression of the said at least one gene in a
comparator tissue;wherein decreased expression of said at least one gene
in the scar of interest compared to expression of said at least one gene
in the comparator tissue indicates that the scar of interest comprises a
keloid.

[0025]In a second aspect of the invention there is provided a kit for
diagnosing a scar of interest as keloid or non-keloid, the kit
comprising:

i) at least one probe capable of binding specifically to a target molecule
representative of expression in the scar of interest of at least one gene
selected from the group set out in Table 1; andii) reference material
able to indicate the level of expression of said at least one gene in
comparator tissue.

[0026]It is preferred that the methods and kits of the invention to be
used for in vitro diagnosis of a scar of interest as keloid or
non-keloid.

[0027]Although the methods and kits of the invention are most suitable for
use in diagnosis of human scars as keloid or non-keloid, they may also be
useful in diagnosing similar conditions in non-human animals, such as
"proud flesh" in horses.

[0028]The present invention is based on the identification by the
inventors of a number of genes the decreased expression of which is
diagnostic of keloid tissue. The inventors have found that comparison of
the expression of one or more of these genes in a scar of interest with
the expression occurring in a comparator tissue allows an accurate and
rapid diagnosis as to whether the tissue is keloid or non-keloid.
Identity of the scar of interest as keloid is indicated by a decrease in
gene expression as compared to expression in the comparator tissue
sample, whereas unchanged or increased expression in the scar of interest
as compared to the comparator indicates that the scar of interest is
non-keloid tissue.

[0029]The finding that decreased expression of the genes identified in
Table 1 (i.e. the group comprising Gene Identification No. 1 to Gene
Identification No. 590) may be used to diagnose whether a scar of
interest is keloid or non-keloid is surprising, since although the
expression of certain genes (such as those encoding VEGF, IGF1 and PAI1)
has been linked to keloid tissue, the genes set out in Table 1 had never
previously been identified as being associated with keloids, let alone as
diagnostic of keloid scars.

[0030]In practicing the invention (whether by use of the methods, kits or
arrays of the invention), expression of a selected gene (or genes) in a
sample representative of gene expression in the scar of interest is
compared with expression of the same gene (or genes) in a suitable
comparator tissue. This comparison of expression of the selected gene (or
genes) enables diagnosis of the scar of interest as keloid or non-keloid.
If there is decreased expression of the selected gene (or genes) in the
sample representative of gene expression in the scar of interest, as
compared to in the comparator sample, then this indicates that the scar
of interest comprises keloid tissue. If, on the other hand, there is no
decrease in expression of the selected gene (or genes) in the sample
representative of expression in the scar of interest (or, indeed, if
there is an increase in expression of these genes), this indicates that
the scar of interest does not comprise keloid tissue.

[0031]In general expression of selected genes in the scar of interest will
be investigated by analysis of target molecules representative of gene
expression. Suitable investigation may involve the analysis for presence
or absence of such target molecules in a sample (qualitative analysis of
gene expression, as discussed further elsewhere in the specification), or
analysis of the relative abundance of target molecules in a sample (which
may provide quantitative information as to gene expression, as considered
in more detail elsewhere in the specification).

[0032]Gene expression in the comparator tissue may be represented by
tissues or tissue extracts containing suitable target molecules, or may
alternatively be represented by data setting out details of the gene
expression levels in the comparator. The identification, isolation and
analysis of suitable target molecules is discussed further elsewhere in
the specification, as is the provision of information representative of
gene expression in comparator tissue samples.

[0033]A comparator tissue, for the purposes of the present disclosure, is
a tissue with which gene expression in a scar of interest can be
compared, in order to allow diagnosis of the scar of interest as keloid
or non-keloid. Specifically, the expression in the scar of interest of at
least one gene set out in Table 1 is compared with expression of the same
gene (or genes) in the comparator tissue.

[0034]A number of different tissue types may serve as suitable comparator
tissues for use in accordance with the present invention. Suitable
comparator tissues include normal skin. For the present purposes normal
skin may be considered to be skin other than in a keloid scar, and
preferably to be unscarred and unwounded skin.

[0035]Alternatively a comparator tissue suitable for use in accordance
with the present invention may be tissue from a known keloid. For
instance a suitable comparator tissue for use in accordance with the
invention may comprise tissue from the skin adjacent to a known keloid
(also referred to in the present specification as "extra-keloid
comparator tissue"). Alternatively, a suitable comparator tissue may
comprise tissue from the area at the periphery of a known keloid (also
referred to herein as "peri-keloid comparator tissue"). In a further
alternative, a suitable comparator tissue may comprise tissue from the
interior part of a known keloid (also referred to as "intra-keloid
comparator tissue").

[0036]A "comparator sample" for the purposes of the present invention
comprises any sample (such as a tissue extract, or the like, as
considered elsewhere in the specification) that provides information as
to the expression of a selected gene in the comparator tissue from which
the comparator sample is derived.

[0037]Although the inventors have found that any of the genes represented
by the group of genes set out in Table 1 may be used in accordance with
the present invention, the inventors have further found that certain
subsets of these genes have particular diagnostic value. These subsets
are identified and considered in more detail below.

[0038]The inventors have noted that expression of certain genes set out in
Table 1 varies between different areas of a keloid lesion. This
information may be used to further refine diagnosis in accordance with
the present invention (whether by methods, kits, or arrays of the
invention).

[0039]The inventors have also found that preferred genes that may be
investigated in the methods and kits of the invention may be selected
with reference to their biological function.

[0040]A sample of interest, representative of a scar of interest to be
diagnosed, may be further characterised with reference to the location
from which it is derived in the scar. The inventors have found that
characterisation of a sample of interest on this basis improves the
efficacy of diagnoses undertaken in accordance with the invention.
Samples of interest may be characterised as peri-lesional (which is to
say samples taken from the periphery of a lesion comprising a scar of
interest) and intra-lesional (those samples taken from the interior of a
lesion comprising a scar of interest).

[0041]Genes from Table 1 that may be used in the diagnosis of
peri-lesional samples of interest are set out in Table 2. It is a
preferred embodiment that diagnosis in accordance with the invention
(whether using the methods, kits or arrays of the invention) may be
performed on the basis of comparison of one or more of the genes set out
in Table 2.

[0042]Genes from Table 1 that may be used in the diagnosis of
intra-lesional samples of interest are set out in Table 20. It is a
preferred embodiment that diagnosis in accordance with the invention
(whether using the methods, kits or arrays of the invention) may be
performed on the basis of comparison of one or more of the genes set out
in Table 20.

[0043]As set out above, comparator tissues suitable for use in diagnosis
in accordance with the invention may also be characterised with reference
to their source, as normal skin comparators; extra-keloid comparators;
peri-keloid comparators; or intra-keloid comparators. The inventors have
found that diagnosis in accordance with the invention may be improved by
comparison of gene expression in a sample of interest characterised with
reference to their location in a scar of interest, with gene expression
of a comparator characterised in the manner set out above.

[0044]Thus it may be preferred that gene expression in a peri-lesional
sample of interest is compared with gene expression in a normal skin
comparator. Examples of suitable genes, expression of which may be
compared between such samples in order to provide a diagnosis, are set
out in Table 3. These genes may be further characterised with reference
to their biological function. Accordingly those genes set out in Table 4
represent genes associated with cell motility, whereas those set out in
Table 5 are associated with cell adhesion, the genes set out in Table 6
are associated with inflammation, and the genes set out in Table 7 are
associated with the development of new blood vessels (particularly with
angiogenesis).

[0045]Alternatively or additionally, it may be preferred that gene
expression in a peri-lesional sample of interest is compared with gene
expression in an extra-keloid comparator. Examples of suitable genes,
expression of which may be compared between such samples in order to
provide a diagnosis, are set out in Table 8. These genes may be further
characterised with reference to their biological function. Accordingly
those genes set out in Table 9 represent genes associated with cell
motility, whereas those set out in Table 10 are associated with cell
adhesion, the genes set out in Table 11 are associated with inflammation,
and the genes set out in Table 12 are associated with the development of
new blood vessels (particularly with angiogenesis).

[0046]Alternatively or additionally, it may be preferred that gene
expression in a peri-lesional sample of interest is compared with gene
expression in a peri-keloid comparator. Examples of suitable genes,
expression of which may be compared between such samples in order to
provide a diagnosis, are set out in Table 13. It will be appreciated that
diagnosis on the basis of such comparisons will involve the gene
expression in a tissue of interest and a comparator that are at different
time-points in the healing process. Information regarding the time-points
to be used is provided in Table 13. The genes set out in Table 13 may
also be further characterised with reference to their biological
function. Accordingly those genes set out in Table 14 are associated with
cell adhesion, the genes set out in Table 15 are associated with
inflammation, and the genes set out in Table 16 are associated with the
development of new blood vessels (particularly with angiogenesis).

[0047]Alternatively or additionally, it may be preferred that gene
expression in a peri-lesional sample of interest is compared with gene
expression in an intra-keloid comparator. Examples of suitable genes,
expression of which may be compared between such samples in order to
provide a diagnosis, are set out in Table 17. These genes may be further
characterised with reference to their biological function. Accordingly
those genes set out in Table 18 represent genes associated with cell
motility, whereas those set out in Table 19 are associated with
inflammation.

[0048]Alternatively or additionally, it may be preferred that gene
expression in an intra-lesional sample of interest is compared with gene
expression in a normal skin comparator. Examples of suitable genes,
expression of which may be compared between such samples in order to
provide a diagnosis, are set out in Table 21.

[0049]Alternatively or additionally, it may be preferred that gene
expression in an intra-lesional sample of interest is compared with gene
expression in an extra-keloid comparator. Examples of suitable genes,
expression of which may be compared between such samples in order to
provide a diagnosis, are set out in Table 22. These genes may be further
characterised with reference to their biological function. Accordingly
those genes set out in Table 23 represent genes associated with cell
motility.

[0050]Alternatively or additionally, it may be preferred that gene
expression in an intra-lesional sample of interest is compared with gene
expression in a peri-keloid comparator. Examples of suitable genes,
expression of which may be compared between such samples in order to
provide a diagnosis, are set out in Table 24. These genes may be further
characterised with reference to their biological function. Accordingly
those genes set out in Table 25 are associated with cell adhesion.

[0051]Alternatively or additionally, it may be preferred that gene
expression in an intra-lesional sample of interest is compared with gene
expression in an intra-keloid comparator. Examples of suitable genes,
expression of which may be compared between such samples in order to
provide a diagnosis, are set out in Table 26. It will be appreciated that
diagnosis on the basis of such comparisons will involve the gene
expression in a tissue of interest and a comparator that are at different
time-points in the healing process. Information regarding the time-points
to be used is provided in Table 26. The genes set out in Table 26 may
also be further characterised with reference to their biological
function. Accordingly those genes set out in Table 27 are associated with
inflammation, and the genes set out in Table 28 are associated with the
development of new blood vessels (particularly with angiogenesis).

[0052]It may be preferred that diagnosis in accordance with the present
invention, whether carried out using the methods, kits or arrays of the
invention, utilise comparison of one or more gene selected independently
from one or more of the Tables 2 to 28.

[0053]A skilled person wishing to undertake a diagnosis in accordance with
the invention may consider the nature of a sample that is available from
a scar of interest, consider the nature of a comparator sample that is
available, and thereby select appropriate genes expression of which may
be compared with reference to the considerations set out above.

[0054]It is particularly preferred that the methods, kits or arrays of the
invention may involve the comparison of genes selected from two or more
of Tables 2 to 28. For example, preferred methods, kits or arrays may
involve the comparison of at least one gene selected from each of two of
Tables 2 to 28, more preferred methods, kits or arrays may involve
comparison of at least one gene selected from each of three of Tables 2
to 28, even more preferred methods, kits or arrays may involve comparison
of at least one gene selected from each of four of Tables 2 to 28, and
most preferred methods, kits or arrays may involve comparison of at least
one gene selected from each of five or more of Tables 2 to 28.

[0055]Diagnosis of a scar of interest as keloid or non-keloid in
accordance with the present invention may be effected by comparing the
expression in a sample representative of gene expression in the scar of
interest with expression in a comparator sample of one gene selected from
Table 1, however, it is preferred to utilise multiple genes from Table 1.
Thus it may be preferred that diagnosis in accordance with the present
invention may be effected by comparing the expression of up to five genes
selected from Table 1. It is particularly preferred that diagnosis in
accordance with the present invention is effected by comparing the
expression of 5, 6, 7, 8, 9 or 10 genes selected from Table 1. Diagnosis
may be effected by comparing expression of up to 20 or 50 genes selected
from Table 1. Diagnosis in accordance with the present invention may be
effected by comparing the expression of up to 100, 200, 300, 400 or even
up to 500, genes selected from Table 1. Indeed it may in certain cases be
preferred that diagnosis of a scar of interest as keloid or non-keloid in
accordance with the present invention is effected by comparing the
expression of 500 or more genes selected from Table 1. If so desired, a
diagnosis can be made using any or all of the 590 genes represented in
Table 1.

[0056]A scar of interest in the context of the present invention may be
any scar for which it is desired to diagnose whether the scar comprises
keloid or non-keloid tissue. It will be appreciated that dermal scars
constitute preferred examples of suitable scars of interest. The ability
to distinguish between keloids and other forms of severe or pathological
scarring, such as hypertrophic scars, is of notable value. Such
differentiation may allow the selection of clinical treatment that is
appropriate to the type of scarring diagnosed. Accordingly, the use of
the methods and kits of the present invention in effecting diagnosis of
excessive or pathological dermal scars represents a particularly
preferred example of their use.

[0057]A scar of interest may preferably be derived from an individual
believed to be at elevated risk of keloid formation. Examples of such
individuals include patients with a history of keloid formation,
individuals of the African Continental Ancestry Group or individuals of
the Asian Continental Ancestry Group.

[0058]Suitable scars of interest may be derived from individuals who have
suffered injury to the skin. In particular these may include individuals
suffering injury at a site where there is an elevated risk of keloid
formation. Examples of such sites may typically include areas of high
skin tension, such as the chest, back, shoulders, or neck. However,
relevant sites may also include areas, such as the earlobes, that are
common sites of keloid formation, although not subject to high skin
tension.

[0059]Diagnosis using the methods, kits, and arrays of the invention may
be useful in diagnosing scars of interest from patients who have
experienced skin wounding, as well as in diagnosing scars of interest
from patients who have experienced skin trauma.

[0060]For the purposes of the present invention "skin wounding" may be
considered to comprise conditions or clinical situations in which partial
or total penetration of the skin occurs, and also those in which partial
or total destruction of one or more layers of the skin occurs. For
example, wounds may include puncture wounds, incisional wounds,
excisional wounds and partial or full thickness skin grafts (including
both donor and recipient sites). Such wounds may be associated with
surgical procedures or accidental injuries. Wounds may also include burn
or scald injuries, resulting from exposure of the skin to substances at
high or low temperatures sufficient to cause damage to the skin. Chemical
"burns", such as those caused by exposure of the skin to acid or alkali,
and cosmetic procedures such as dermabrasion or exfoliation (included
so-called "chemical peels" and "laser peels") may also give rise to
tissues for which it is wished to produce a diagnosis in accordance with
the present invention.

[0061]For the purposes of the present invention "skin trauma" may be taken
as referring to injuries that damage, but do not penetrate, the skin.
Illustrative examples of injuries that may be considered as skin trauma
include crush injuries to the skin, as well other "blunt" injuries.

[0062]Although the preceding paragraphs provide examples of individuals,
or of scars of interest, that may particularly benefit from diagnosis in
accordance with the present invention it will be appreciated that the
methods, kits and arrays of the invention may be beneficially used in
diagnosis of any scar of interest, particularly those that may be
believed to be keloid scars. Generally tissues that are believed possibly
to be keloid scars will be those that display one or more characteristics
selected from the following group: an elevated profile compared to the
surrounding skin; a lesion growing beyond its original boundaries; a
lesion at the site of an earlier skin wound or trauma; hypo- or
hyper-pigmentation compared to the surrounding skin.

[0063]Samples representative of gene expression in a scar of interest that
may be used in accordance with the present invention encompass any sample
that may provide information as to genes being expressed by the scar of
interest.

[0064]Any suitable sample derived from the scar of interest may be used.
Preferred sample include biopsies and, if available, samples of wound
tissue, wound fluid, wound aspirates or wound exudates. Preferably such
biopsies may be of a sort selected to reduce the level of injury
inflicted to the patient, and thereby limit damage to those found to
reduce the risk of (further) keloid formation. Such techniques may, for
example, make use of needle biopsies in order to reduce the level of
injury occurring. Any of the sample types discussed above may be used in
diagnosis, in accordance with the invention, of the scar of interest from
which the sample in question is derived.

[0065]Suitable samples may include tissue sections such as histological or
frozen sections. Methods by which such sections may be prepared in such a
way as to be able to provide information representative of gene
expression in the scar of interest from which the section is derived will
be well known to those skilled in the art, and should be selected with
reference to the technique that it is intended to use when investigating
gene expression.

[0066]Although the use of samples comprising a portion of the scar of
interest is contemplated, it may generally be preferred that the sample
representative of gene expression comprise a suitable extract taken from
the scar of interest, said extract being capable of investigation to
provide information regarding gene expression in the scar of interest.
Suitable protocols which may be used for the production of tissue
extracts capable of providing information regarding gene expression in a
scar of interest will be well known to those skilled in the art.
Preferred protocols may be selected with reference to the manner in which
gene expression is to be investigated. Illustrative examples of protocols
that may be used to produce tissue extracts representative of gene
expression in a scar of interest are discussed below.

[0067]Suitable comparator samples, for use in accordance with methods,
kits or arrays of the invention, may be selected with reference to the
scar of interest in respect of which diagnosis is to be performed.
Preferably the comparator tissue will be as well matched as possible to
the scar of interest (matching may consider tissue type, tissue site,
etc.). Sources and examples of suitable comparator samples will be
apparent to those skilled in the art and include those derived from
individuals that are not subject to keloid formation, as well as samples
from keloid formers selected with reference to their location relative to
a known keloid (i.e. non-keloid tissue, extra-keloid tissue,
peri-lesional tissue, or intra-lesional tissue). It will be recognised
that the skin constitutes a preferred source of comparator samples.

[0068]Suitable comparator samples may include portions of non-keloid
tissues or organs including target molecules representative of gene
expression (in which case the tissue should be preserved in such a manner
that information regarding the expression of genes in the tissue may be
extracted from the tissue, for example by analysis of the target
molecules). Alternatively, suitable comparator samples may comprise
tissue extracts incorporating extracted and/or isolated target molecules
(such as mRNA or cDNA) that are representative of gene expression in the
comparator sample. Relevant information regarding gene expression in
comparator samples may also be provided in the form of data derived from
such samples, as considered elsewhere in the specification.

[0069]Comparator samples from which information relating to the expression
of selected genes may be derived include tissue samples and tissue
extracts as considered herein with reference to samples derived from the
scar of interest. For example, such information may be derived directly
from a tissue or organ sample constituting the comparator sample, or from
an extract capable of providing information regarding gene expression in
the selected control sample. The expression of the selected gene, or
genes, (selected from the group of genes set out in Table 1) in
comparator samples of this type may be investigated using the methods
described herein in connection with the investigation of gene expression
in the scar of interest.

[0070]Although tissue or organ samples constituting comparator samples, or
extracts from such samples, may be used directly as the source of
information regarding gene expression in the comparator sample (as
discussed elsewhere in the specification), it will generally be preferred
that information regarding the expression of the selected gene (or genes)
in the comparator sample be provided in the form of reference data. Such
reference data may be provided in the form of tables indicative of gene
expression in the chosen comparator tissue. Alternatively, the reference
data may be supplied in the form of computer software containing
retrievable information indicative of gene expression in the chosen
comparator tissue. The reference data may, for example, be provided in
the form of an algorithm enabling comparison of expression of at least
one selected gene (or genes) in the scar of interest with expression of
the same gene (or genes) in the comparator tissue sample.

[0071]In a preferred embodiment of the invention, a diagnosis as to
whether the scar of interest is keloid or non-keloid may be delivered
automatically on inputting results representative of expression of
selected genes in the scar of interest into a predictive algorithm that
has been trained upon data representative of gene expression in a
suitable comparator sample. Well-established and commonly used
classification systems include, but are not limited to, K-Nearest
Neighbours, Centroid Classification, Linear Discriminant Analysis, Neural
Networks and Support Vector Machines available, for example, in the
Partek Genomics Suite software package (Partek Inc.).

[0072]A suitable sample representative of gene expression in a scar of
interest or comparator sample may provide qualitatitive and/or
quantitative information regarding gene expression. For the purpose of
the present invention qualitative information regarding gene expression
is to be considered to be information that provides identification as to
genes expressed in a scar of interest or comparator sample, without
providing information as to the relative amounts of expression (save as
to whether a particular gene is, or is not, expressed). It will be
appreciated that in some situations qualitative information may allow a
sufficient comparison between expression in the scar of interest and the
comparator sample to allow a diagnosis as to whether the scar of interest
is keloid or non-keloid.

[0073]Qualitative information may be particularly suitable for diagnoses
that are based on decreased expression of genes of Table 1 that are
normally expressed in comparator samples, but are not expressed at all in
keloids. In such cases the lack of expression of the gene the scar of
interest will be sufficient to indicate an elevated risk of keloid
formation. Examples include those genes identified by Gene Identification
Numbers 2, 3 and 4 and it may be a preferred embodiment of the invention
to investigate expression of these genes. The inventors have found that
these genes may be used as the basis of a diagnosis when their expression
is compared (quantitatively or qualitatively) between an intra-lesional
scar of interest and a normal skin comparator.

[0074]It will, however, generally be preferred to use a sample capable of
providing quantitative information regarding gene expression in the scar
of interest or comparator sample. Such information allows ready
comparison between the levels of expression in the scar of interest and
the levels of expression in the comparator sample. For the purposes of
the present invention quantitative information relating to gene
expression may be taken to refer to either absolute or relative
quantification. Methods by which absolute or relative quantitation may be
achieved are discussed further below.

[0075]Samples representative of gene expression in the scar of interest or
comparator sample will generally contain target molecules that are
directly or indirectly representative of gene expression. Suitable
samples may be provided in the form of tissue samples containing such
target molecules, or, preferably as tissue extracts. A tissue extract
representative of gene expression in a scar of interest will generally
contain isolated target molecules that are representative of gene
expression in the tissue from which the extract is obtained.

[0076]Suitable techniques by which tissue samples or tissue extracts may
be obtained and prepared in order that they may provide information as to
gene expression may be selected with reference to the type of target
molecule that is to be employed. Examples of appropriate techniques that
may be used will be readily apparent to the skilled person, however
guidance as to suitable techniques is also provided elsewhere in the
specification.

[0077]It will be appreciated that protein target molecules represent
target molecules that are particularly amenable to direct detection. Such
direct detection may provide qualitative or quantitative information as
to the amount of the protein present in the scar of interest or
comparator sample, thereby allowing comparison of expression.

[0079]The presence or absence of target molecules in a tissue sample or
extract will generally be detected using suitable probe molecules
(although there may be some instances, such as those discussed above,
where presence or absence of a target molecule may be determined directly
without the need for a probe). Such detection will provide information as
to gene expression, and thereby allow comparison between gene expression
occurring in the scar of interest and expression occurring in the
comparator sample. Diagnosis in accordance with the invention may be
carried out based on such comparisons.

[0080]Probes will generally be capable of binding specifically to target
molecules directly or indirectly representative of gene expression in the
scar of interest or comparator sample. Binding of such probes may then be
assessed and correlated with gene expression to allow an effective
diagnostic comparison between gene expression in the scar of interest and
in the comparator. Suitable probes that may be used in the methods, kits
and arrays of the invention are discussed elsewhere in the specification.

[0081]Target molecules suitable for use in the methods, kits and arrays of
the invention are molecules representative of gene expression either
directly or indirectly, as considered in greater detail below. Target
molecules may include mRNA gene transcripts, as well as natural and
artificial products of such transcripts (e.g. proteins or cDNA
respectively). It will be appreciated that samples for use in accordance
with the present invention should be processed in a manner selected with
reference to the nature of the target molecule that is to be used.
Suitable protocols for processing of tissues to yield samples containing
usable target molecules are discussed further below.

[0082]Suitable target molecules may comprise the direct products of gene
expression. Such direct products of gene expression may, for example,
comprise one or more gene transcripts representative of gene expression.
The use of mRNA gene transcripts as target molecules allowing comparison
of gene expression in the scar of interest with expression in the
comparator sample is a preferred embodiment of the invention.

[0083]Alternatively, a sample representative of gene expression in the
scar of interest or comparator sample may comprise target molecules that
are indirectly representative of gene expression. Examples of such
targets indirectly representative of gene expression may include natural
products (such as proteins) that are produced on translation of a gene
transcript, as well as artificial products generated from gene
transcripts. Preferred examples of artificial target molecules generated
from gene transcripts include cDNA and cRNA, either of which may be
generated using well known protocols or commercially available kits or
reagents.

[0084]For example, in a preferred embodiment, RNA representative of gene
expression in a scar of interest or comparator sample may be isolated
through a process of lysing cells taken from a suitable sample (which may
be achieved using a commercially available lysis buffer such as that
produced by Qiagen Ltd.) followed by centrifugation of the lysate using a
commercially available nucleic acid separation column (such as the RNeasy
midi spin column produced by Qiagen Ltd). Other methods for RNA
extraction include variations on the phenol and guanidine isothiocyanate
method of Chomczynski, P. and Sacchi, N. (1987) Analytical Biochemistry
162, 156. "Single Step Method of RNA Isolation by Acid Guanidinium
Thiocyanate-Phenol-Chloroform Extraction." RNA obtained in this manner
may constitute a suitable target molecule itself, or may serve as a
template for the production of target molecules representative of gene
expression.

[0085]It may be preferred that RNA derived from a scar of interest or
comparator sample may be used as substrate for cDNA synthesis, for
example using the Superscript System (Invitrogen Corp.). The resulting
cDNA may then be converted to biotinylated cRNA (for instance using the
BioArray RNA Transcript labelling kit from Enzo Life Sciences Inc.) and
this cRNA purified from the reaction mixture (for instance using an
RNeasy mini kit from Qiagen Ltd).

[0087]The disclosures of the documents set out in the preceding paragraphs
are incorporated by reference, insofar as they describe methods that may
useful to the skilled person in practising the present invention.

[0088]In the event that expression of one or more genes from Table 1 in a
comparator sample is to be investigated via processing of a tissue or
organ sample constituting the comparator sample, or by processing of a
tissue extract representative of gene expression in the comparator
sample, for example to isolate suitable target molecules, it is preferred
that such processing is conducted using the same methods used to process
the sample from the scar of interest. Such parallel processing of samples
from both the scar of interest and comparator tissue allows a greater
degree of confidence that comparisons of gene expression in these tissues
will be normalised relative to one another (since any artifacts
associated with the selected method by which tissue is processed and gene
expression investigated will be applied to both the scar of interest and
comparator samples).

[0089]Furthermore, the parallel processing of the comparator sample in
this manner provides an "internal control" that will allow the
practitioner to confirm that processing has occurred successfully. Since
the practitioner will be aware that the selected one or more genes from
Table 1 that have been selected for comparison of expression are normally
expressed by comparator tissues, the practitioner will be able to
discount any instances of processing (for investigation of gene
expression) which give rise to assays indicating that expression of these
internal controls cannot be detected (since these results will likely be
as a result of a processing error leading to artificially low readings).
Such results may otherwise give rise to an incorrect assessment that the
scar of interest comprises keloid tissue (since the same artificial
decrease in assessed expression would be noted in respect of the selected
gene or genes from Table 1).

[0090]Samples representative of gene expression in a scar of interest, or
a comparator tissue, may be manipulated prior to effecting comparison of
gene expression. Such manipulation may, for example, be designed to make
comparison of expression easier, or to increase the information made
available by the comparison. Examples of suitable ways in which such
samples may be manipulated are considered below.

[0091]Preferably the methods or kits of the invention will provide means
by which the expression data relating to the scar of interest and
comparator tissue may be "normalised" with respect to one another.
Normalisation ensures that comparisons being made are "like for like",
and suitable parameters for use in normalisation are well known to those
skilled in the art. Purely by way of illustration, normalisation may be
effected with reference to cell numbers in the samples to be compared;
and/or total protein content of samples to be compared; and/or total
nucleic acid content of samples to be compared; and/or expression level
of one or more genes the expression of which does not change between
keloid and non-keloid tissues. Alternatively or additionally, a suitable
control may involve assessing expression of one or more genes known to be
expressed in keloids. Detection of the expression of such genes (in
combination with the reduced expression of one or more of the genes set
out in Table 1) will provide a suitable control against which gene
expression can be referenced. Suitable examples of such genes are
considered elsewhere in the specification.

[0092]The inventors have found that preferred samples representative of
gene expression for use in accordance with the present invention are
those samples comprising nucleic acid target molecules representative of
gene expression. For the purposes of the present invention a nucleic acid
target is a nucleic acid the presence or absence of which is to be
detected, or the amount of which present is to be quantified. Such
detection or quantification will allow a diagnostic comparison of
expression to be effected. A target nucleic acid may preferably have a
sequence that is complementary to the nucleic acid sequence of a
corresponding probe directed to the target. A nucleic acid target in
accordance with the present invention may encompass both a specific
subsequence of a larger nucleic acid to which a probe is directed or,
alternatively, the overall sequence (e.g. complete mRNA transcript) whose
expression level it is desired to detect. Suitable nucleic acid targets
may include both RNAs and DNAs, and encompass both naturally occurring
and artificial nucleic acids.

[0093]It will be understood that target nucleic acids suitable for use in
accordance with the invention need not comprise "full length" nucleic
acids (e.g. full length gene transcripts), but need merely comprise a
sufficient length to allow specific binding of probe molecules.

[0094]It will be understood that "nucleic acids" or "nucleic acid
molecules" for the purposes of the present invention refer to a
deoxyribonucleotide or ribonucleotide polymers in either single- or
double-stranded form. Furthermore, unless the context requires otherwise,
these terms should be taken to encompass known analogues of natural
nucleotides that can function in a similar manner to naturally occurring
nucleotides.

[0095]mRNA constitutes a preferred form of target molecule that may be
used in the methods and kits of the invention. mRNA gene transcripts are
directly representative of gene expression in the scar of interest or
comparator sample.

[0096]It will be recognised that mRNA, representative of gene expression,
may be found directly in a scar of interest or comparator sample, without
the need for mRNA extraction or purification. For example, mRNA present
in, and representative of gene expression in, a scar of interest or
comparator sample may be investigated using appropriately fixed sections
or biopsies of such a tissue. The use of samples of this kind may provide
benefits in terms of the rapidity with which comparisons of expression
can be made, as well as the relatively cheap and simple tissue processing
that may be used to produce the sample. In situ hybridisation techniques
represent preferred methods by which gene expression may be investigated
and compared in tissues samples of this kind. Techniques, for the
processing of scars of interest that maintain the availability of RNA
representative of gene expression in the scar of interest or comparator
sample are well known to those of skill in the art.

[0097]However, techniques by which mRNAs representative of gene expression
in a scar of interest or comparator sample may be extracted and collected
are well known to those skilled in the art, and the inventors have found
that such techniques may be advantageously employed in accordance with
the present invention. Samples comprising extracted mRNA from a scar of
interest or comparator sample may be preferred for use in the methods and
kits of the invention, since such extracts tend to be more readily
investigated than is the case for samples comprising the original
tissues. For example, suitable target molecules allowing for comparison
of gene expression may comprise the total RNA isolated from a sample of
the scar of interest, or a sample of comparator tissue.

[0098]Furthermore, extracted RNA may be readily amplified to produce an
enlarged mRNA sample capable of yielding increased information on gene
expression in the scar of interest or comparator sample. Suitable
examples of techniques for the extraction and amplification of mRNA
populations are well known, and are considered in more detail below.

[0099]By way of example, methods of isolation and purification of nucleic
acids to produce nucleic acid targets suitable for use in accordance with
the invention are described in detail in Chapter 3 of Laboratory
Techniques in Biochemistry and Molecular Biology:

[0101]In a preferred method, the total nucleic acid may be isolated from a
given sample using, for example, an acid guanidinium-phenol-chloroform
extraction method.

[0102]In the event that it is desired to amplify the nucleic acid targets
prior to investigation and comparison of gene expression it may be
preferred to use a method that maintains or controls for the relative
frequencies of the amplified nucleic acids in the scar of interest or
control tissue from which the sample is derived.

[0103]Suitable methods of "quantitative" amplification are well known to
those of skill in the art. One well known example, quantitative PCR
involves simultaneously co-amplifying a control sequence whose quantities
are known to be unchanged between comparator samples and those from the
scar of interest. This provides an internal standard that may be used to
calibrate the PCR reaction.

[0104]In addition to the methods outlined above, the skilled person will
appreciate that any technology coupling the amplification of
gene-transcript specific product to the generation of a signal may also
be suitable for quantitation. A preferred example employs convenient
improvements to the polymerase chain reaction (U.S. Pat. No. 4,683,195
and 4683202) that have rendered it suitable for the exact quantitation of
specific mRNA transcripts by incorporating an initial reverse
transcription of mRNA to cDNA. Further key improvements enable the
measurement of accumulating PCR products in real-time as the reaction
progresses. Examples of suitable technologies using fluorescent resonance
energy transfer to generate a quantitative gene-specific signal include
Taqman (U.S. Pat. No. 5,210,015 and 5487972), molecular beacons
(WO-95/13399) and scorpions (US2005/0164219). The parallel quantitation
of multiple transcripts is possible via the use of different fluorescent
moieties for each gene target.

[0106]In a particularly preferred embodiment, the mRNA transcripts from a
tissue representative of gene expression in a scar of interest or
comparator sample may be reverse transcribed with a reverse transcriptase
and a promoter consisting of oligo dT and a sequence encoding the phage
T7 promoter to provide single stranded DNA template. The second DNA
strand is polymerized using a DNA polymerase. After synthesis of
double-stranded cDNA, T7 RNA polymerase is added and RNA is transcribed
from the cDNA template. Successive rounds of transcription from each
single cDNA template results in amplified RNA. Methods of in vitro
polymerization are well known to those of skill in the art (see, e.g.,
Sambrook, supra.) and this particular method is described in detail by
Van Gelder, et al., Proc. Natl. Acad. Sci. USA, 87: 1663-1667 (1990) who
demonstrate that in vitro amplification according to this method
preserves the relative frequencies of the various RNA transcripts.
Moreover, Eberwine et al. Proc. Natl. Acad. Sci. USA, 89: 3010-3014
(1992) provide a protocol that uses two rounds of amplification via in
vitro transcription to achieve greater than 106 fold amplification
of the original starting material, thereby permitting expression
monitoring even when only a small sample of the scar of interest is
available.

[0107]It will be appreciated by one of skill in the art that the direct
transcription method described above leads to the production of antisense
RNA (aRNA) targets. In such cases probes, such as oligonucleotide probes,
to be used to investigate and compare gene expression should be chosen to
be complementary to sequences or sub-sequences of the antisense nucleic
acids.

[0108]The skilled person will further appreciate that artificial nucleic
acid molecules may also be used in the comparison of gene expression.
Examples of artificial target molecules suitable for use in accordance
with the present invention include cDNAs made by reverse transcription of
mRNA or second strand cDNA or RNA (cRNA) transcribed from a double
stranded cDNA intermediate. Methods for the production of cDNAs and cRNAs
are well documented in the art, and will be known to the skilled person,
and indeed kits and reagents suitable for their production are readily
commercially available.

[0109]For the purposes of the present invention, a sample that is
"representative" of gene expression in a scar of interest is to be
considered to encompass any sample providing information as to the
expression of genes in the scar of interest. For example, a
representative sample may provide information as to all the genes
expressed in the scar of interest, and preferably the relative levels of
expression of said genes.

[0110]In a preferred embodiment, a representative sample is one in which
the concentration of target molecules is proportional to the
concentration of mRNA gene transcripts of the gene (or genes) expression
of which, in the scar of interest, is to be compared to comparators.
While it is preferred that the proportionality be relatively strict
(e.g., a doubling in the number of mRNA gene transcript occurring in the
scar of interest leading to a doubling in the number of corresponding
target molecules present in the sample), the skilled person will
appreciate that the proportionality can be more relaxed and even
non-linear. For example, an assay where a five fold difference in
concentration of the mRNA gene transcripts in the scar of interest
results in a three to six fold difference in the concentration of target
molecules in the representative sample is sufficient for most purposes.

[0111]In the event that more precise quantification is required, serial
dilutions of "standard" target molecules can be used to prepare
calibration curves according to methods well known to those skilled in
the art. More preferably quantitation of target molecules will be
relative and normalised with respect to each other and/or "housekeeping"
genes whose expression levels are not increased in keloid forming as
compared to non-keloid forming tissues. Examples of such genes include
exportin 7 (XPO7), Cleavage and Polyadenylation Specific Factor 4, 30 kDa
(CPSF4), F-box only protein 7 (FBXO7), ADP-ribosylation factor 1 (ARF1),
signal sequence receptor, beta (SSR2) and methionine-tRNA synthetase
(MARS).

[0112]It will, of course, be appreciated that in the case of a qualitative
sample or samples (where simple detection of the presence or absence of
gene expression is desired) no such elaborate control or calibration is
required.

[0113]Although it may be preferred in many instances that the
representative sample provides information as to all genes expressed in
the scar of interest or comparator sample, a suitable representative
sample may alternatively provide information relating to the expression
of only a sub-set of the total number of genes undergoing expression.

[0114]In many cases it may be preferred to assess the degree of gene
expression in a scar of interest or comparator sample using probe
molecules capable of indicating the presence of target molecules
(representative of one or more of the genes set out in Table 1) in the
relevant sample.

[0115]The use of target molecules and probes in methods, kits or assays in
accordance with the present invention may confer increased sensitivity on
the methods of the invention. This may lead to an increased ability to
discriminate between otherwise small differences between expression in
the scar of interest and expression in the comparator sample. This will
have appreciable benefits on diagnosis in accordance with the invention.

[0116]Generally, suitable probes for use in the present invention will
bind to their target molecules, and thereby allow detection of the target
molecule (this detection being indicative of expression of the gene
selected from Table 1 represented by the target molecule).

[0117]It may be preferred that probes for use in accordance with the
invention allow replication of the target molecules (suitably in
combination with the probe molecule). Replication in this manner produces
a greater number of target molecules, and thus allows further binding of
the labelled probe. In turn, the increased amount of labelled probe thus
bound amplifies the detectable signal indicative of gene expression.

[0118]Probes for use in the methods and kits of the invention may be
selected with reference to the product (direct or indirect) of gene
expression to be investigated. Examples of suitable probes include
oligonucleotide probes, antibodies, aptamers, and binding proteins or
small molecules having suitable specificity.

[0119]Oligonucleotide probes constitute preferred probes suitable for use
in accordance with the methods and kits of the invention. The generation
of suitable oligonucleotide probes is well known to those skilled in the
art (Oligonucleotide synthesis: Methods and Applications, Piet Herdewijn
(ed) Humana Press (2004).). Oligonucleotide and modified oligonucleotides
are commercially available from numerous companies.

[0120]An oligonucleotide is a single-stranded nucleic acid ranging in
length from 2 to about 500 nucleotide bases, preferably from about 5 to
about 50 nucleotides, more preferably from about 10 to about 40
nucleotides and most preferably from about 15 to about 40 nucleotides in
length. Suitable hybridization methods, conditions, times, fluid volumes,
and suitable methods by which hybridisation of oligonucleotide probes may
be detected are as described elsewhere in the present specification.

[0121]For the purposes of the present invention an oligonucleotide probe
may be taken to comprise an oligonucleotide capable of hybridising
specifically to a target nucleic acid of complementary sequence through
one or more types of chemical bond. Such binding may usually occur
through complementary base pairing, and usually through hydrogen bond
formation. Suitable oligonucleotide probes may include natural (ie., A,
G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In
addition, a linkage other than a phosphodiester bond may be used to join
the bases in an oligonucleotide probe, so long as this variation does not
interfere with hybridisation of the oligonucleotide probe to its target.
Thus, oligonucleotide probes suitable for use in the methods and kits of
the invention may be peptide nucleic acids in which the constituent bases
are joined by peptide bonds rather than phosphodiester linkages.

[0122]The phrase "hybridising specifically to" as used herein refers to
the binding, duplexing, or hybridising of an oligonucleotide probe
preferentially to a particular target nucleotide sequence under stringent
conditions when that sequence is present in a complex mixture (such as
total cellular DNA or RNA). Preferably a probe may bind, duplex or
hybridise only to the particular target molecule.

[0123]The term "stringent conditions" refers to conditions under which a
probe will hybridise to its target subsequence, but minimally to other
sequences. Preferably a probe may hybridise to no sequences other than
its target under stringent conditions. Stringent conditions are
sequence-dependent and will be different in different circumstances.
Longer sequences hybridise specifically at higher temperatures.

[0124]In general, stringent conditions may be selected to be about
5° C. lower than the thermal melting point (Tm) for the specific
sequence at a defined ionic strength and pH. The Tm is the temperature
(under defined ionic strength, pH, and nucleic acid concentration) at
which 50% of the oligonucleotide probes complementary to a target nucleic
acid hybridise to the target nucleic acid at equilibrium. As the target
nucleic acids will generally be present in excess, at Tm, 50% of the
probes are occupied at equilibrium. By way of example, stringent
conditions will be those in which the salt concentration is at least
about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30° C. for short probes
(e.g., 10 to 50 nucleotides). Stringent conditions may also be achieved
with the addition of destabilizing agents such as formamide.

[0125]Considerations for the design and selection of probes suitable for
use with antisense nucleic acid targets (aRNA) have been discussed above.
In the case that the nucleic acid targets comprise sense nucleic acids,
suitable oligonucleotide probes may be selected to be complementary to
sequences or sub-sequences of the sense nucleic acids. In the case of
nucleic acid targets that are double stranded, suitable probes may be of
either sense as the nucleic acid targets will provide both sense and
antisense strands.

[0126]Antibodies suitable for use in the methods or kits of the invention
may be used to detect target molecules, such as proteins, that represent
gene expression in a scar of interest.

[0127]Antibodies that may be used to investigate gene expression in
accordance with the methods and kits of the present invention include
monoclonal antibodies and polyclonal antibodies, as well as fragments of
such antibodies, including, but not limited to, Fab or F(ab')hd 2, and Fv
fragments.

[0128]Methods suitable for the generation and/or identification of
antibodies capable of binding specifically to a given target are well
known to those skilled in the art. In general suitable antibodies may be
generated by the use of the isolated target as an immunogen. This
immunogen is administered to a mammalian organism, such as, but not
limited to, a rat, rabbit, goat or mouse, and antibodies elicited as part
of the immune response. Generally antibodies will be used in the context
of the methods and kits of the invention to bind to protein products of
gene expression. Suitable immunogens may include the full-length protein
to be investigated, or an antigenic peptide fragment thereof.

[0129]Monoclonal antibodies can be produced by hybridomas, immortalized
cell lines capable of secreting a specific monoclonal antibody. The
immortalized cell lines can be created in vitro by fusing two different
cell types, usually lymphocytes, one of which is a tumour cell.

[0130]Aptamers are nucleic acid molecules that assume a specific,
sequence-dependent shape and bind to specific target ligands based on a
lock-and-key fit between the aptamer and ligand. Typically, aptamers may
comprise either single- or double-stranded DNA molecules (ssDNA or dsDNA)
or single-stranded RNA molecules (ssRNA).

[0131]Aptamers may be used to bind both nucleic acid and non-nucleic acid
targets. Accordingly aptamers are suitable probes for use in the
investigation of gene expression products including RNA, DNA and small
molecules or proteins. Preferably aptamers may be used to investigate
gene expression products having a molecular weight of between 100 and
10,000 Da. ssDNA aptamers may be preferred for use in the investigation
of gene expression products comprising DNA.

[0132]Suitable aptamers may be selected from random sequence pools, from
which specific aptamers may be identified which bind to the selected
target molecules with high affinity. Methods for the production and
selection of aptamers having desired specificity are well known to those
skilled in the art, and include the SELEX (systematic evolution of
ligands by exponential enrichment) process. Briefly, large libraries of
oligonucleotides are produced, allowing the isolation of large amounts of
functional nucleic acids by an iterative process of in vitro selection
and subsequent amplification through polymerase chain reaction.

[0133]The use of aptamers for investigation of gene expression in
accordance with the methods and kits of the invention may be
advantageous, since aptamers have relatively stable shelf lives. Aptamers
suitable for use in the methods and/or kits of the invention may
preferably be stabilized by chemical modifications (for example
2'-NH2 and 2'-F modifications).

[0134]Photoaptamers are a subclass of aptamers incorporating at least one
bromo-deoxyuridine (BrdU) in place of a thymidine (T) nucleotide. The
presence of the BrdU enables photoaptamers to form a specific covalent
crosslink with their target ligands when exposed to ultraviolet light.
Because crosslinking requires both affinity-based binding and close
proximity between a BrdU (at a specific location in the photoaptamer) and
an amino acid (at a specific location in the target ligand),
photoaptamers may be preferred for use in the methods and kits of the
invention when increased specificity of binding with a gene expression
product is required.

[0135]Suitable methods by which gene expression may be compared in
accordance with the present invention may be selected in the light of the
considerations referred to in the preceding pages.

[0136]In general methods for analysis may be selected based on the nature
of a target molecule to be investigated, and suitable selection criteria
may distinguish between nucleic acid and protein target molecules.

[0137]However, as set out above, it may generally be preferred to
investigate and compare gene expression using oligonucleotide probes
capable of binding to nucleic acid target molecules.

[0138]Oligonucleotide probes may be used to detect complementary nucleic
acid sequences (i.e., nucleic acid targets) in a suitable representative
sample. Such complementary binding forms the basis of most techniques in
which oligonucleotides may be used to detect, and thereby allow
comparison of, expression of particular genes. Preferred technologies
permit the parallel quantitation of the expression of multiple genes and
include technologies where amplification and quantitation of species are
coupled in real-time, such as the quantitative reverse transcription PCR
technologies previously described herein, and technologies where
quantitation of amplified species occurs subsequent to amplification,
such as array technologies.

[0139]Array technologies involve the hybridisation of samples,
representative of gene expression within the scar of interest or
comparator sample, with a plurality of oligonucleotide probes wherein
each probe preferentially hybridises to a disclosed gene or genes. Array
technologies provide for the unique identification of specific
oligonucleotide sequences, for example by their physical position (e.g.,
a grid in a two-dimensional array as commercially provided by Affymetrix
Inc.) or by association with another feature (e.g. labelled beads as
commercially provided by Illumina Inc or Luminex Inc). Oligonucleotide
arrays may be synthesised in situ (e.g by light directed synthesis as
commercially provided by Affymetrix Inc) or pre-formed and spotted by
contact or ink-jet technology (as commercially provided by Agilent or
Applied Biosystems). It will be apparent to those skilled in the art that
whole or partial cDNA sequences may also serve as probes for array
technology (as commercially provided by Clontech).

[0140]Oligonucleotide probes may be used in blotting techniques, such as
Southern blotting or northern blotting, to detect and compare gene
expression (for example by means of cDNA or mRNA target molecules
representative of gene expression). Techniques and reagents suitable for
use in Southern or northern blotting techniques will be well known to
those of skill in the art. Briefly, samples comprising DNA (in the case
of Southern blotting) or RNA (in the case of northern blotting) target
molecules are separated according to their ability to penetrate a gel of
a material such as acrylamide or agarose. Penetration of the gel may be
driven by capillary action or by the activity of an electrical field.
Once separation of the target molecules has been achieved these molecules
are transferred to a thin membrane (typically nylon or nitrocellulose)
before being immobilized on the membrane (for example by baking or by
ultraviolet radiation). Gene expression may then be detected and compared
by hybridisation of oligonucleotide probes to the target molecules bound
to the membrane. More details of suitable conditions in which
hybridisation may be effected are provided below, as are examples of
techniques by which hybridisation may be detected.

[0141]In certain circumstances the use of traditional hybridisation
protocols for comparing gene expression may prove problematic. For
example blotting techniques may have difficulty distinguishing between
two or more gene products of approximately the same molecular weight
since such similarly sized products are difficult to separate using gels.
Accordingly, in such circumstances it may be preferred to compare gene
expression using alternative techniques, such as those described below.

[0142]Gene expression in a sample representing gene expression in a scar
of interest may be assessed with reference to global transcript levels
within suitable nucleic acid samples by means of high-density
oligonucleotide array technology. Such technologies make use of arrays in
which oligonucleotide probes are tethered, for example by covalent
attachment, to a solid support. These arrays of oligonucleotide probes
immobilized on solid supports represent preferred components to be used
in the methods and kits of the invention for the comparison of gene
expression. Large numbers of such probes may be attached in this manner
to provide arrays suitable for the comparison of expression of large
numbers of genes selected from those set out in Table 1. Accordingly it
will be recognised that such oligonucleotide arrays may be particularly
preferred in embodiments of the methods or kits of the invention where it
is desired to compare expression of more than one gene selected from
Table 1 in order to effect a diagnosis.

[0143]In a preferred embodiment investigation of gene expression using
oligonucleotide arrays may be effected by hybridisation of
oligonucleotide probes and nucleic acid targets at low stringency
followed by at least one wash at higher stringency. Low stringency
conditions suitable for use in accordance with these embodiments may
comprise a reaction temperature of about 20° C. to about
50° C. (more preferably about 30° C. to about 40°
C., and most preferably about 37° C.) and 6×SSPE-T buffer
(or lower). Suitable hybridisation protocols may include subsequent
washes at progressively increasing stringency until a desired level of
hybridisation specificity is reached. Hybridisation stringency may also
be varied by electronic means, for example as provided by Nanogen Inc.
(Sosnowski R, Heller M J, Tu E, Forster A H, Radtkey R. Active
microelectronic array system for DNA hybridization, genotyping and
pharmacogenomic applications. Psychiatr Genet. 2002 December;
12(4):181-92).

[0144]Suitable techniques for the detection of hybridisation between
oligonucleotide probes and nucleic acid targets are considered further
below.

[0145]The identity of selected oligonucleotide probes incorporated in
arrays may be altered to allow more detailed selection of the genes, the
expression of which is to be compared. For example arrays suitable for
use in the methods or kits of the invention may comprise one or more
oligonucleotide probes selected with reference to the differential
expression of selected genes from Tables 1 to 28 as considered
previously.

[0146]Alternatively, assessment of gene expression in a scar of interest
or comparator sample based on levels of nucleic acids sequences (such as
mRNA or DNA) in a sample representative of gene expression in the scar of
interest or comparator may be undertaken using other suitable techniques
that will be apparent to the skilled person. For example, northern
blotting provides a sensitive method by which levels of mRNA
representative of gene expression in a scar of interest or comparator
sample may be assessed.

[0149]For instance, expression of proteins having enzymatic activity may
be investigated and compared using assays based around activity of the
protein in question. Enzymatic protein extracts (here constituting
samples representative of gene expression in the scar of interest or
comparator sample) may, for example, be incubated with samples comprising
known quantities of the appropriately labelled substrate. The amount of
enzymatic activity, and hence an indication of the level of gene
expression in the scar of interest or comparator sample, may be
determined by the amount of substrate converted by the enzyme.

[0150]Detection of probe or target molecules can be facilitated by
coupling (i.e., physical linking) of such molecules to a detectable
moiety. Alternatively suitable probe or target molecules may be
synthesised such that they incorporate detectable moieties. Techniques
that may be used in the coupling or incorporation of detectable moieties
in probe or target molecules suitable for use in the method, kits or
arrays of the invention are considered below.

[0151]Examples of detectable moieties that may be used in the labelling of
probes or targets suitable for use in accordance with the invention
include any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Suitable detectable moieties include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent materials,
radioactive materials and colorimetric materials. These detectable
moieties are suitable for incorporation in all types of probes or targets
that may be used in the methods or kits of the invention unless indicated
to the contrary.

[0153]Means of detecting such labels are well known to the skilled person.
For example, radiolabels may be detected using photographic film or
scintillation counters; fluorescent markers may be detected using a
photodetector to detect emitted light. Enzymatic labels are typically
detected by providing the enzyme with a substrate and detecting the
reaction product produced by the action of the enzyme on the substrate,
and colorimetric labels are detected by simply visualizing the coloured
label.

[0154]In a preferred embodiment of the invention fluorescently labelled
probes or targets may be scanned and fluorescence detected using a laser
confocal scanner.

[0155]In the case of labelled nucleic acid probes or targets suitable
labelling may take place before, during, or after hybridisation. In a
preferred embodiment, nucleic acid probes or targets for use in the
methods or kits of the invention are labelled before hybridisation.
Fluorescence labels are particularly preferred and, where used,
quantification of the hybridisation of the nucleic acid probes to their
nucleic acid targets is by quantification of fluorescence from the
hybridised fluorescently labelled nucleic acid. More preferably
quantitation may be from a fluorescently labelled reagent that binds a
hapten incorporated into the nucleic acid.

[0156]In a preferred embodiment of the invention analysis of hybridisation
may be achieved using suitable analysis software, such as the Microarray
Analysis Suite (Affymetrix Inc.) and diagnosis automated by use of
classification software (for example Partek Genomics Suite from Partek
Inc).

[0157]Effective quantification may be achieved using a fluorescence
microscope which can be equipped with an automated stage to permit
automatic scanning of the array, and which can be equipped with a data
acquisition system for the automated measurement, recording and
subsequent processing of the fluorescence intensity information. Suitable
arrangements for such automation are conventional and well known to those
skilled in the art.

[0158]In a preferred embodiment, the hybridised nucleic acids are detected
by detecting one or more detectable moieties attached to the nucleic
acids. The detectable moieties may be incorporated by any of a number of
means well known to those of skill in the art.

[0159]However, in a preferred embodiment, such moieties are simultaneously
incorporated during an amplification step in the preparation of the
sample nucleic acids (probes or targets). Thus, for example, polymerase
chain reaction (PCR) using primers or nucleotides labelled with a
detectable moiety will provide an amplification product labelled with
said moiety. In a preferred embodiment, transcription amplification using
a fluorescently labelled nucleotide (e.g. fluorescein-labelled UTP and/or
CTP) incorporates the label into the transcribed nucleic acids.

[0160]Alternatively, a suitable detectable moiety may be added directly to
the original nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc. from
the scar of interest) or to an amplification product after amplification
of the original nucleic acid is completed. Means of attaching labels such
as fluorescent labels to nucleic acids are well known to those skilled in
the art and include, for example nick translation or end-labelling (e.g.
with a labeled RNA) by kinasing of the nucleic acid and subsequent
attachment (ligation) of a nucleic acid linker joining the sample nucleic
acid to a label (such as a suitable fluorophore).

[0161]As set out previously, in addition to the methods and kits described
above, the invention also provides a kit for diagnosing a scar of
interest as keloid or non-keloid, the kit comprising:

i) at least one probe capable of binding specifically to a target molecule
representative of expression in a scar of interest of at least one gene
selected from the group set out in Table 1; andii) reference material
able to indicate the level of expression of said at least one gene in a
comparator sample.

[0162]Preferably kits in accordance with this aspect of the invention may
further comprise assay control material able to indicate that an assay
has been performed correctly. Suitably such assay control material may
include target molecules representative of expression of genes the
expression of which does not vary between keloid and non-keloid tissues.
Suitable examples of such housekeeping genes are considered elsewhere in
the specification, and target molecules representative of expression of
any of these genes may be advantageously provided in the kits of the
invention. The provision of housekeeping genes of this sort in known
quantities may provide a "standard" against which assay results may be
normalised.

[0163]It may be preferred that a kit according to the present invention
further comprises material (such as target molecules) representative of
one or more genes whose expression is increased in keloid tissue. The
provision of such genes may increase the ability to discriminate a
biologically meaningful result from a change in the absolute input
material or a change in the efficiency of any assay process. For example,
lysyl oxidase displays a 3-fold higher expression in keloid tissue than
in non-keloid tissue. Lysyl oxidase is a key enzyme involved in collagen
cross-linking and has previously been shown to be highly expressed in
fibrotic tissue.

[0164]Kits of the invention may further comprise materials for the
preparation of a population of target molecules representative of gene
expression in a scar of interest (or in a comparator tissue). Such
materials may be suitable for the preparation of a population of nucleic
acid target molecules. Alternatively such materials may be suitable for
the preparation of a population of protein target molecules. It may be
preferred that the kits comprise materials for the preparation of a
population of labelled target molecules representative of gene expression
in a scar of interest or comparator tissue.

[0165]It is also preferred that kits of the present invention may further
comprise an algorithm or reference data/material able to indicate that
the level of expression of said at least one gene, selected from the
group set out in Table 1, in the scar of interest is diagnostic that the
scar of interest is keloid tissue.

[0166]The algorithm may be provided in the form of a mathematical model of
the difference in gene expression of said at least one gene, selected
from the group set out in Table 1, between comparator data and data from
scars of interest (such as known patient data). This mathematical model
may then be deployed on gene expression data of said at least one gene,
selected from the group set out in Table 1, from a new patient sample.
The output thus generated will thus provide a diagnosis as to whether a
scar of interest comprises keloid or non-keloid tissue.

[0167]Probes for inclusion in kits in accordance with this second aspect
of the invention may be selected using the same criteria as for the first
aspect of the invention. Suitable probes may be selected from the group
comprising oligonucleotide probes, antibodies, aptamers and specific
binding proteins.

[0168]Kits in accordance with the present invention may preferably
comprise probes capable of binding specifically to target molecules
representative of expression of up to five genes selected from the group
set out in Table 1 (i.e. target molecules representative of the
expression of up to five genes selected from Table 1). It is particularly
preferred that kits of the invention comprise probes capable of binding
5, 6, 7, 8, 9 or 10 such target molecules. Kits may include probes
capable of binding to up to 20 or up to 50 genes selected from those set
out in Table 1. Suitable kits may comprise probes capable of binding to
up to 100, 200, 300, 400 or 500 such target molecules. Indeed, kits of
the invention may comprise probes capable of binding specifically to 500
or more target molecules, and may even comprise probes capable of binding
specifically to targets representative of expression of all 590 of the
genes set out in Table 1.

[0169]A kit of the invention will comprise probes capable of binding to
target molecules representative of expression of at least one gene
selected from Table 1, and/or probes capable of binding to target
molecules representative of expression of at least one gene selected from
Table 2, and/or probes capable of binding to target molecules
representative of expression of at least one gene selected from Table 3,
and/or probes capable of binding to target molecules representative of
expression of at least one gene selected from Table 8, and/or probes
capable of binding to target molecules representative of expression of at
least one gene selected from Table 13, and/or probes capable of binding
to target molecules representative of expression of at least one gene
selected from Table 17, and/or probes capable of binding to target
molecules representative of expression of at least one gene selected from
Table 20, and/or probes capable of binding to target molecules
representative of expression of at least one gene selected from Table 22,
and/or probes capable of binding to target molecules representative of
expression of at least one gene selected from Table 24, and/or probes
capable of binding to target molecules representative of expression of at
least one gene selected from Table 26.

[0170]Kits of the invention may include probes capable of binding to
target molecules representative of gene expression of any of the genes
set out in any one of (or any combination of) Tables 2 to 28.

[0171]The probes provided in the kits of the invention may preferably be
labelled probes. Labelled probes may comprise any detectable moiety
considered in connection with the first aspect of the invention.
Preferred labelled probes may be chosen from the group comprising
haptens, fluorescently labelled probes, radioactively labelled probes and
enzymatically labelled probes.

[0172]The reference material provided in kits of the invention may
comprise a library of nucleic acid targets representative of expression
in an appropriate comparator sample of one or more genes selected from
the group of genes set out in Table 1.

[0173]In a preferred embodiment the reference material may comprise
recorded information regarding the level of expression of one or more
genes selected from the group of genes set out in Table 1 in keloid and
non-keloid tissue.

[0174]In a most preferred example the reference data may be used to create
an algorithm which may deliver a diagnosis based upon the level of
expression of one or more genes selected from the group of genes set out
in Table 1.

[0175]Oligonucleotide probes provided in kits of the invention, may
preferably be provided in the form of an oligonucleotide array as
considered elsewhere in the specification.

[0176]It will be appreciated from the preceding pages that the use of
oligonucleotide arrays is particularly useful in effecting a diagnosis in
accordance with the present invention as to whether a scar of interest is
keloid or non-keloid tissue.

[0177]Accordingly, in a third aspect of the invention there is provided an
array of oligonucleotide probes, characterised in that at least 7.0% of
the oligonucleotides probes present in the array are representative of
genes selected from the group of genes set out in Table 1.

[0178]The invention also provides an array comprising immobilized antibody
probes capable of binding specifically to molecules representative of
expression of one or more of the group of genes set out in Table 1.
Furthermore, the invention also provides an array comprising a nylon
substrate to which are adhered nucleic acid probes representative of
genes selected from the group of genes set out in Table 1. The nucleic
acid probes may preferably be cDNA molecules.

[0179]Although a planar array surface is preferred, the array may be
fabricated on a surface of virtually any shape or even a multiplicity of
surfaces. In a further example a suitable array may be fabricated on the
surface of a library of addressable beads, in which each bead displays a
known nucleic acid sequence. Alternatively, a suitable array may be
fabricated on the surface of a nylon substrate, typically a woven or
non-woven nylon membrane.

[0180]It will be appreciated that arrays in accordance with the present
invention can be used to compare the expression of a large number of
genes set out in Table 1 simultaneously (and indeed to compare
simultaneous expression of such genes), and that this gives rise to
significant advantages in reduced labour, cost and time. Furthermore, the
comparison of expression levels of multiple genes allows a greater degree
of confidence in diagnoses that may be effected in accordance with the
invention.

[0181]An array in accordance with the present invention may comprise up to
five probes specific for genes selected from the group set out in Table
1. Preferably an array may comprise 5, 6, 7, 8, 9 or 10 probes specific
for genes selected from the group set out in Table 1. Arrays may comprise
probes specific for up to 20 or up to 50 genes selected from the group
set out in Table 1. Suitable arrays may comprise up to 100, up to 200, up
to 300, up to 400 or up to 500 probes specific genes selected from the
group set out in Table 1. Indeed, suitable arrays may comprise probes
specific for 500 or more of the genes set out in Table 1, and may even
comprise probes specific for all 590 genes set out in Table 1. It will be
appreciated that each of the probes should be specific for a different
selected gene, and that more than one copy of each probe may be provided.

[0182]Arrays of the invention may comprise probes capable of binding to a
target representative of expression of at least one gene selected from
the group set out in Table 2, and/or probes capable of binding to a
target representative of expression of at least one gene selected from
the group set out in Table 3, and/or probes capable of binding to a
target representative of expression of at least one gene selected from
the group set out in Table 8, and/or probes capable of binding to a
target representative of expression of at least one gene selected from
the group set out in Table 13, and/or probes capable of binding to a
target representative of expression of at least one gene selected from
the group set out in Table 17, and/or probes capable of binding to a
target representative of expression of at least one gene selected from
the group set out in Table 20, and/or probes capable of binding to a
target representative of expression of at least one gene selected from
the group set out in Table 22, and/or probes capable of binding to a
target representative of expression of at least one gene selected from
the group set out in Table 24, and/or probes capable of binding to a
target representative of expression of at least one gene selected from
the group set out in Table 26.

[0183]An array according to the present invention may comprise probes
capable of binding to targets representative of the expression of one or
more genes selected from any one of, or any combination of, Tables 1 to
28.

[0184]It is preferred that an array according to the present invention may
further comprise one or more genes whose expression is increased in
keloid tissue. The provision of such genes may increase the ability to
discriminate a biologically meaningful result from a change in the
absolute input material or a change in the efficiency of any assay
process. For example, lysyl oxidase displays a 3-fold higher expression
in keloid tissue. Lysyl oxidase is a key enzyme involved in collagen
cross-linking and has previously been shown to be highly expressed in
fibrotic tissue.

[0185]The methods, kits and arrays of the invention may also make use of
one or more "housekeeping genes" to provide a control by which the
efficiency of any assay may be assessed. These housekeeping genes may be
provided in the kits of the invention, or on the arrays of the invention.
Suitable housekeeping genes will be those that are either invariant
between keloid and non-keloid tissue or show no association with keloid
formation. Examples of genes that display invariant expression in both
keloid and non-keloid (comparator) biopsy samples include exportin 7
(XPO7), Cleavage and Polyadenylation Specific Factor 4, 30 kDa (CPSF4),
F-box only protein 7 (FBXO7), ADP-ribosylation factor 1 (ARF1), signal
sequence receptor, beta (SSR2) and methionine-tRNA synthetase (MARS).

[0186]Oligonucleotide arrays in accordance with the invention may be
synthesized by any suitable technique known in the art. A preferred
technique that may be used in the synthesis of such arrays is
light-directed very large scaled immobilized polymer synthesis (VLSIPS),
which has previously been described in a number of publications (Lipshutz
R J, Fodor S P, Gingeras T R, Lockhart D J. High density synthetic
oligonucleotide arrays. Nat Genet. 1999 January; 21(1 Suppl):20-4; Jacobs
J W, Fodor S P. Combinatorial chemistry--applications of light-directed
chemical synthesis. Trends Biotechnol. 1994 January; 12(1):19-26).

[0187]An oligonucleotide array in accordance with the invention may allow
comparison of hybridisation, and thereby gene expression, to be carried
out in extremely small fluid volumes (e.g., 250 μl or less, more
preferably 100 μl or less, and most preferably 10 μl or less). This
confers a number of advantages. In small volumes, hybridization may
proceed very rapidly. In addition, hybridization conditions are extremely
uniform throughout the sample, and the hybridization format is amenable
to automated processing.

[0188]The skilled person will appreciate that diagnosis in accordance with
the present invention (whether carried out using the methods, kits or
arrays of the invention) may be useful in assessing the efficacy of a
treatment employed to alleviate or cure keloid scarring. A keloid in
which a treatment is producing a beneficial effect may be identified by
virtue of its ability to alleviate the reduction of expression observed
in respect of the genes set out in any of Tables 1 to 28.

[0189]A treatment that renders expression of one or more genes selected
from Table 1 within a treated keloid more similar to the expression of
said gene (or genes) found in a normal skin comparator should be viewed
as having a beneficial effect on the keloid being treated. In the event
that expression in a treated keloid is not rendered more similar to the
expression found in a normal skin comparator, the treatment may be viewed
as not beneficial to the keloid scar in question. In such cases it may be
wished to adopt an alternative treatment strategy, and optionally to
subsequently assess the effectiveness of the alternative strategy in the
same manner.

Table Legends

[0190]Genes the expression of which may be investigated in accordance with
the present invention are set out in the accompanying Tables. These
Tables provide, in respect of each gene, a Gene Identification Number; a
Public Identifier and Data Source (by which the skilled person may
identify the gene in question and obtain further information regarding
its sequence); the Gene Name; a Probe ID (setting out details of at least
one probe that may be used to investigate expression of the gene in
question); details of tissues that may be used in comparing expression of
the gene in question; as well as details of the Fold Change in expression
and P value derived from comparisons conducted as described in the
Experimental Results section.

[0191]Table 1: Genes that diagnose a keloid scar. All genes are highly
statistically significant with p-values less than 0.01.

[0192]Table 2: Genes that may be used in the diagnosis of a peri-lesional
sample of a scar of interest as a keloid or non-keloid scar.

[0193]Table 3: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and a normal skin comparator
to diagnose the scar of interest as a keloid or non-keloid scar.

[0194]Table 4: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and a normal skin comparator
to diagnose the scar of interest as a keloid or non-keloid scar. Genes
identified in this table encode proteins with cell motility functionality
in accordance with the Gene Ontology classification (GO:0006928).

[0195]Table 5: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and a normal skin comparator
to diagnose the scar of interest as a keloid or non-keloid scar. Genes
identified in this table encode proteins with cell adhesion functionality
in accordance with the Gene Ontology classification (GO:0007155).

[0196]Table 6: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and a normal skin comparator
to diagnose the scar of interest as a keloid or non-keloid scar. Genes
identified in this table encode proteins with inflammation functionality
in accordance with the Gene Ontology classification (GO:0006954).

[0197]Table 7: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and a normal skin comparator
to diagnose the scar of interest as a keloid or non-keloid scar. Genes
identified in this table encode proteins with angiogenesis functionality
in accordance with the Gene Ontology classification (GO:0001525).

[0198]Table 8: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and an extra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar.

[0199]Table 9: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and an extra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar. Genes identified in this table encode proteins with cell motility
functionality in accordance with the Gene Ontology classification
(GO:0006928). Table 10: Genes the expression of which may be compared
between a peri-lesional sample from a scar of interest and an
extra-keloid comparator to diagnose the scar of interest as a keloid or
non-keloid scar. Genes identified in this table encode proteins with cell
adhesion functionality in accordance with the Gene Ontology
classification (GO:0007155).

[0200]Table 11: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and an extra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar. Genes identified in this table encode proteins with inflammation
functionality in accordance with the Gene Ontology classification
(GO:0006954).

[0201]Table 12: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and an extra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar. Genes identified in this table encode proteins with angiogenesis
functionality in accordance with the Gene Ontology classification
(GO:0001525).

[0202]Table 13: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and a peri-keloid comparator
to diagnose the scar of interest as a keloid or non-keloid scar.

[0203]Table 14: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and a peri-keloid comparator
to diagnose the scar of interest as a keloid or non-keloid scar. Genes
identified in this table encode proteins with cell adhesion functionality
in accordance with the Gene Ontology classification (GO:0007155).

[0204]Table 15: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and a peri-keloid comparator
to diagnose the scar of interest as a keloid or non-keloid scar. Genes
identified in this table encode proteins with inflammation functionality
in accordance with the Gene Ontology classification (GO:0006954).

[0205]Table 16: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and a peri-keloid comparator
to diagnose the scar of interest as a keloid or non-keloid scar. Genes
identified in this table encode proteins with angiogenesis functionality
in accordance with the Gene Ontology classification (GO:0001525).

[0206]Table 17: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and an intra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar.

[0207]Table 18: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and an intra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar. Genes identified in this table encode proteins with cell motility
functionality in accordance with the Gene Ontology classification
(GO:0006928).

[0208]Table 19: Genes the expression of which may be compared between a
peri-lesional sample from a scar of interest and an intra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar. Genes identified in this table encode proteins with inflammation
functionality in accordance with the Gene Ontology classification
(GO:0006954).

[0209]Table 20: Genes that may be used in the diagnosis of an
intra-lesional sample of a scar of interest as a keloid or non-keloid
scar.

[0210]Table 21: Genes the expression of which may be compared between an
intra-lesional sample from a scar of interest and a normal skin
comparator to diagnose the scar of interest as a keloid or non-keloid
scar.

[0211]Table 22: Genes the expression of which may be compared between an
intra-lesional sample from a scar of interest and an extra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar.

[0212]Table 23: Genes the expression of which may be compared between an
intra-lesional sample from a scar of interest and an extra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar. Genes identified in this table encode proteins with cell motility
functionality in accordance with the Gene Ontology classification
(GO:0006928).

[0213]Table 24: Genes the expression of which may be compared between an
intra-lesional sample from a scar of interest and a peri-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar.

[0214]Table 25: Genes the expression of which may be compared between an
intra-lesional sample from a scar of interest and a peri-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar. Genes identified in this table encode proteins with cell adhesion
functionality in accordance with the Gene Ontology classification
(GO:00071 55).

[0215]Table 26: Genes the expression of which may be compared between an
intra-lesional sample from a scar of interest and an intra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar.

[0216]Table 27: Genes the expression of which may be compared between an
intra-lesional sample from a scar of interest and an intra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar. Genes identified in this table encode proteins with inflammation
functionality in accordance with the Gene Ontology classification
(GO:0006954).

[0217]Table 28: Genes the expression of which may be compared between an
intra-lesional sample from a scar of interest and an intra-keloid
comparator to diagnose the scar of interest as a keloid or non-keloid
scar. Genes identified in this table encode proteins with angiogenesis
functionality in accordance with the Gene Ontology classification
(GO:0001 525).

[0218]The invention will now be further described with reference to the
following Experimental Results.

EXPERIMENTAL RESULTS

[0219]The suitability of the genes set out in Table 1 for use in the
diagnosis of scars of interest as keloid or non-keloid is illustrated by
the following study. In this study expression of the genes set out in
Table 1 was compared between samples taken from known keloid tissues and
suitably matched comparator tissues.

1.1 Diagnosis of Keloid Tissue.

[0220]Twenty patients of the African Continental Ancestry Group who had
keloids that had been established for at least one year provided keloid
samples for use in the present study. Only keloids for which a full
medical history could be established were included. The age of the scar,
a thorough review of the scar history and examination by a clinician,
ensured that the scar had been correctly diagnosed as keloidal and not
hypertrophic.

[0221]Three African Continental Ancestry Group subjects with no history of
keloid formation provided control comparator tissue ("normal comparator")
for use in the study described herein.

1.2 Tissue Collection.

[0222]Keloids were sampled using ellipsoid excisions perpendicular to the
keloid margin and the resulting biopsies were sectioned to provide
samples comprising skin surrounding the keloid lesion (extra-keloid
tissue), the peripheral portion of the keloid lesion (peri-lesional
tissue), or the interior part of the keloid lesion (intra-lesional
tissue). Since these tissues were selected from stringently diagnosed
examples of keloids they provided a suitable experimental example to test
the diagnostic capacity of the genes set out in Table 1.

[0223]Extra-keloid tissue collected in these procedures was used as a
comparator tissue (extra-keloid comparator) for use in the following
studies. Skin tissue from non-keloid forming individuals was also
biopsied in a similar manner to provide relevant non-keloid comparator
tissues.

[0224]Once collected, the biopsy sections were immersed in RNA Later
solution (Ambion) and stored at -80° C. until later analysis of
gene expression.

1.3 Preparation of Samples Representative of Gene Expression in Tissue.

[0225]Peri-lesional, intra-lesional and extra-lesional samples from keloid
formers and skin samples from non-keloid formers were disrupted using a
Diax (G-10) homogeniser in the presence of proprietary Qiagen lysis
buffer, and the lysate produced then incubated with proteinase K at
55° C. for 20 minutes.

[0226]Following incubation the mixture was separated by centrifugation,
and RNA present purified using a RNeasy midi spin column (Qiagen Ltd).

1.4 Production of Nucleic Acid Targets.

[0227]10 μg total RNA was used as substrate for cDNA synthesis using
the Superscript System (Invitrogen Corp.). The resulting cDNA was then
converted to biotinylated cRNA target molecules using the BioArray RNA
Transcript labelling Kit (Enzo Life Sciences Inc.). The cRNA target
molecules were subsequently purified from the reaction mixture using a
RNeasy mini kit (Qiagen Ltd). 20 μg cRNA was fragmented for array
hybridisation.

1.5 Comparison of Gene Expression.

[0228]Fragmented cRNA target molecules representative of gene expression
in peri- and intra-lesional keloid tissues and in extra-keloid and
non-keloid comparator tissues were hybridised to oligonucleotide arrays
comprising oligonucleotide probes representing the genes set out in Table
1. Standard Affymetrix protocols (Affymetrix Inc) were used to effect
hybridisation. The hybridised arrays were stained with
streptavidin-phycoerythrin and then scanned using a laser confocal
scanner to generate fluorescence intensities.

[0229]All arrays were normalised to a target intensity of 1000, and signal
values and detection P-values were calculated using the Microarray
Analysis Suite version 5.0 software. Data sets passing quality control
were imported into the Spotfire analysis suite for comparison of
expression with that in comparator tissues.

[0230]Signal values were transformed to log 2 scale and t-tests comparing
the gene expression in samples representative of keloids with expression
in comparators were performed on the log 2 transformed data. Mean signal
values were calculated for each sample group and fold changes were
calculated from these mean values.

1.6 Results.

[0231]T-tests comparing expression of the genes set out in Table 1 in
keloid tissues (peri- and intra-lesional tissues) with expression of the
same genes in comparator tissues all had a t-test p-value of less than
0.01. This confirms that the expression of each and all of the genes set
out in Table 1 are highly significantly decreased in keloid tissue as
opposed to comparators.

[0232]These results clearly illustrate that decreased expression in a
sample from a scar of interest of one or more genes from the group set
out in Table 1, as compared to expression of the same gene or genes in a
comparator sample, provides a clear diagnosis that the scar of interest
is a keloid tissue.